LOCUS       sequence-147283-        5196 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector for cell-free expression with N-term 
            myc-tag mCherry tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5196)
  AUTHORS   Gagoski D, Mureev S, Giles N, Johnston W, Dahmer-Heath M, Skalamera 
            D, Gonda TJ, Alexandrov K
  TITLE     Gateway-compatible vectors for high-throughput protein expression in
            pro- and eukaryotic cell-free systems.
  JOURNAL   J Biotechnol. 2015 Feb 10;195:1-7. doi: 
            10.1016/j.jbiotec.2014.12.006. Epub 2014 Dec 19.
  PUBMED    25529348
REFERENCE   2  (bases 1 to 5196)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J 
            Biotechnol."; date: "2015-02-10"; volume: "195:1-7. doi"; pages: " 
            10.1016/j.jbiotec.2014.12.006. Epub 2014 Dec 19"
COMMENT     Sequence Label: pCellFree_G07
FEATURES             Location/Qualifiers
     source          1..5196
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(84..100)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(84..100)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(97..119)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    108..124
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(132..138,139..156,157..162))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    177..198
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(315..332)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(486..1074)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(566..585)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1245..2036,2037..2105))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERTRQIAEIGAS
                     LIKHW"
     primer_bind     1868..1887
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2106..2210)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2242..2261
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2242..2260
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2395..2424
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2434..2457
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2464..3165
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="SKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQ
                     TAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFED
                     GGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGE
                     IKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEG
                     RHSTGGMDELYK"
     primer_bind     complement(2603..2621)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2871..2890
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3166..3189
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    3190..3314
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(3361..3666)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(3463..3482)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(4008..4667)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4582..4601
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4668..4770)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      join(5156..5196,1..6)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gttttttgct gaaaggagga cagctgatga ttgtcatgct tgccatctgt tttcttgcaa
       61 ggtcagagga attcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
      121 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
      181 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
      241 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
      301 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
      361 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
      421 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
      481 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
      541 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
      601 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
      661 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
      721 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
      781 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
      841 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
      901 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
      961 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     1021 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaggat
     1081 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     1141 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     1201 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     1261 agtgaggcac ctatctcagc gatctgtcta gttcgttcat ccatagttgc ctgactcccc
     1321 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     1381 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     1441 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     1501 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     1561 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     1621 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     1681 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     1741 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     1801 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     1861 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     1921 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     1981 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     2041 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     2101 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     2161 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     2221 cgaaaagtgc cacctgacgt ctaatacgac tcactatagg gacatcttaa gtttatttta
     2281 ttttatttta ttttatttta ttttatttta ttttatttta ttttatttta tttaaccatg
     2341 acagtaatgt ataaagtctg taaagacatt aaacacgtaa gtgaaaccat gggcgagcag
     2401 aagctgatct cggaggagga tctgacctct ggtcaccacc atcaccacca tcaccacggc
     2461 acaagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
     2521 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     2581 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     2641 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     2701 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     2761 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     2821 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     2881 cagaagaaga ctatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     2941 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
     3001 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     3061 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     3121 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagctgga ggtgctcttc
     3181 cagggtccga caagtttgta caaaaaagct gaacgagaaa cgtaaaatga tataaatatc
     3241 aatatattaa attagatttt gcataaaaaa cagactacat aatactgtaa aacacaacat
     3301 atccagtcac tatggggccc gtcgacctgc agactggctg tgtataaggg agcctgacat
     3361 ttatattccc cagaacatca ggttaatggc gtttttgatg tcattttcgc ggtggctgag
     3421 atcagccact tcttccccga taacggagac cggcacactg gccatatcgg tggtcatcat
     3481 gcgccagctt tcatccccga tatgcaccac cgggtaaagt tcacgggaga ctttatctga
     3541 cagcagacgt gcactggcca gggggatcac catccgtcgc ccgggcgtgt caataatatc
     3601 actctgtaca tccacaaaca gacgataacg gctctctctt ttataggtgt aaaccttaaa
     3661 ctgcatttca ccagtccctg ttctcgtcag caaaagagcc gttcatttca ataaaccggg
     3721 cgacctcagc catcccttcc tgattttccg ctttccagcg ttcggcacgc agacgacggg
     3781 cttcattctg catggttgtg cttaccagac cggagatatt gacatcatat atgccttgag
     3841 caactgatag ctgtcgctgt caactgtcac tgtaatacgc tgcttcatag cacacctctt
     3901 tttgacatac ttcgggtata catatcagta tatattctta taccgcaaaa atcagcgcgc
     3961 aaatacgcat actgttatct ggcttttagt aagccggatc cacgcgttta cgccccgccc
     4021 tgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg gaagccatca
     4081 cagacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa
     4141 tatttgccca tggtgaaaac gggggcgaag aagttgtcca tattggccac gtttaaatca
     4201 aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc aataaaccct
     4261 ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga
     4321 aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc agtttgctca
     4381 tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc gtctttcatt
     4441 gccatacgga attccggatg agcattcatc aggcgggcaa gaatgtgaat aaaggccgga
     4501 taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg
     4561 gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc tttacgatgc
     4621 cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt agcttcctta
     4681 gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatcttat ttcattatgg
     4741 tgaaagttgg aacctcttac gtgccgatca acgtctcatt ttcgccaaaa gttggcccag
     4801 ggcttcccgg tatcaacagg gacaccagga tttatttatt ctgcgaagtg atcttccgtc
     4861 acaggtattt attcggcgca aagtgcgtcg ggtgatgctg ccaacttagc aagctttctt
     4921 gtacaaagtg gtgtaagcgg ccgccctcct cctcctttct tgttcctttc acgtcgcctt
     4981 ctcggttgta gctggcagac gacgagtctt acttttacgt gtacttctct atagatgatg
     5041 tatgatctct ctgcatgcgt gttcgtgcat gtgtccgtgt gttgggtacg cgtggtaccc
     5101 tgcaggaagg aagctgagtt ggctgctgcc accgctgagc aataactagt aattactagc
     5161 ataacccctt ggggcctcta aacgggtctt gagggg
//
